Current models predict that NF-:B controlled genes are regulated combinatorially by signal specific factors. This proposal focuses on a factor we identified termed SIMPL. SIMPL is a TNF1 regulated p65 specific co- activator. Our hypothesis is that in response to TNF1, SIMPL is directed to the nucleus, where it couples p65 to the transcriptional machinery, thereby inducing the expression of a subset of genes that regulate the innate immune response. The molecular details of SIMPL regulation by TNF1 and the mechanism by which SIMPL exerts its co-activator function are lacking. We propose that SIMPL contains three regions: a nuclear localization signal whose activity is regulated by phosphorylation, a region required for p65 complex formation and a region required for complexing with Mediator and/or RNA pol II. The latter two regions may also be regulated by phosphorylation. Our first hypothesis is that phosphorylation of SIMPL directs it to the nucleus. Our second hypothesis is that SIMPL functions as a co-activator by serving as an adapter that allows p65 to interact with Mediator and/or RNA pol II. We will test these hypotheses by examining if SIMPL cluster charge and phosphorylation mutants undergo nuclear accumulation and whether they are found in p65, Mediator and/or RNA pol II containing complexes. Examining the activity of SIMPL mutants in SIMPL-/- and p65-/- fibroblasts will enable us to mechanistically dissect and define regions in SIMPL important for its co-activator function. qRT-PCR and ChIP assays will be performed to characterize genes known to require SIMPL for expression. Elucidating how SIMPL activity is regulated and how it regulates TNF1 dependent NF-:B controlled gene expression will provide a paradigm for discovering other signal specific NF-:B co-activators. Transcriptional co-activators like SIMPL are potential drug targets for highly fatal states like septic shock. SIMPL-/- mice, like TNF1-/- and TNF RI-/- mice have defects in hematopoietic cells which play an integral role in the regulation and mediation of the innate immune response. Our discovery of SIMPL, its role in the control of TNF1 dependent p65 activity, our generation of SIMPL-/- mice and the availability of fibroblasts from SIMPL-/- mice place us in a unique position to address at the molecular level signal specific control of NF-:B dependent transcription in a physiologically relevant context. PUBLIC HEALTH RELEVANCE: Through the activation of NF-:B controlled genes TNF1 coordinates activation and resolution of an inflammatory response. TNF1 dependent co-activators like SIMPL that regulate NF-kB activity are potential drug targets for highly fatal states like septic shock and other diseases like like myelodysplastic syndrome, Crohn's disease, rheumatoid arthritis that are linked to dysregulated TNF1 activity.